This invention relates to methods for inhibition of growth of transformed cells, and treatment and diagnosis of diseases and conditions related to ErbB-4 expression.
The epidermal growth factor (EGF) receptors have been implicated in human cancer more frequently than any other family of growth factor receptors. The EGF receptor gene is often amplified or overexpressed in squamous cell carcinoma and glioblastomas [Jenkins et al. (1989) Cancer Genet. Cytogenet. 39:253]. Similarly, ErbB-4 is overexpressed in adenocarcinomas of the stomach, breast and ovary.
The epidermal growth factor receptor (EGFR/ErbB) family is a group of tyrosine kinases that is frequently overexpressed in a variety of carcinomas [Gullick, W. J. (1991) Br. Med. Bull. 47:87-98; Hynes, N. E. and Stern, D. F. (1994) Biochem. Biophys. Acta 1198:165-184; Lemoine, N. R. et al. (1992) Br. J. Cancer 66:1116-1121]. This class I subfamily of receptors is comprised of four members: EGFR [Xu, Y. H. et al. (1984) Nature 309:806-810], HER2/ErbB-2/neu [Schechter, A. L. et al. (1984) Nature 312:513-516], HER3/ErbB-3 [Kraus, M. H. et al. Proc. Natl. Acad. Sci. USA 86:9193-9197; Plowman, G. D. et al. (1990) Proc. Natl. Acad. Sci. USA 87:4905-4909], and HER4/ErbB-4 [Plowman, G. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750]. Data from numerous laboratories suggest that the EGFR family members may play a complex role in signaling [Wada, T. et al. (1990) Cell 61:1339-1347; Goldman, R. et al. (1990) Biochemistry 29:11024-11028; Caraway, K. L. and Cantley L. C. (1994) Cell 78:5-8]. Most human breast cancer cells express more than one of the EGF family receptors, and different combinations of receptors can heterodimerize or homodimerize. These receptor interactions lead to the activation of multiple signaling pathways and contribute to the pathogenicity and tumorigenicity of breast cancer [Earp, S. H. et al. (1995) Breast Cancer Resarch and Treatment]. 
A number of growth factors, classified as EGF-like ligands, have been identified that bind and stimulate the kinase activity of EGF-family receptors. EGF, transforming growth factor xcex1 (TGFxcex1), amphiregulin (AR), heparin-binding EGF(HB-EGF), and betacellulin (BTC) have been described as specific for EGFR [Savage, C. R. et al. (1972) J. Biol. Chem. 241:7612-7621; Marquardt, H. et al. (1983) Science 223:1079-1082; Shoyab, M. et al. (1989) Science 243:1079-1082; Higashiyama, S. et al. (1991) Science 251: 936-939; shing, Y. et al. (1993) Science 259:1604-1607]. Several differentially spliced variants, named heregulin (HRG) also known as neuregulin (NRG), or neu differentiation factor (NDF) [Holmes, W. E. et al. (1992) Science 256:1205-1210; Wen, D. et al. (1992) Cell 69:559-572], acetylcholine-receptor inducing activity (ARIA) [Falls, D. G. et al. (1993) Cell 72:801-815], glial growth factor (GGF) [Marchionni, M. A. et al. (1993) Nature (London)362: 312-318] and gp30 [Lupu, R. et al. (1990) Science 249:1552-1555], were initially identified as candidate neu ligands by their ability to induce neu tyrosine phosphorylation [Peles, E. and Yarden, Y. (1993) Bioassays 15:815-824]. However, recent results demonstrate that ErbB-3 and ErbB-4 are primary receptors for heregulin [Plowman, G. D. et al. (1993) Nature 366:473-475; Carraway, K. L. III et al. (1994) J. Biol. Chem. 269: 14303-14306]. Activation of ErbB-2 by HRG is thought to occur through transphosphorylation resulting from heterodimerization with either ErbB-3 or ErbB-4 [Tzahar, E. et al. (1994) J. Biol. Chem. 269:40:25226-25223; Peles, E. et al. (1993) EMBO J. 12:961-971; Sliwkowski, M. X. et al. (1994) J. Biol. Chem. 269: 14661-14665]. Most recently, betacellulin was also shown to activate the ErbB-4 receptor in a Ba/F3 system [Riesell, D. J. et al. (1996) Oncogene 12: 245-353].
Amplification and/or overexpression of EGFR and ErbB-2 are clearly important factors in neoplastic transformation of breast epithelium [Jardines, L. et al. (1993) Pathobiology 61:268-282]. Elevated ErbB-4 levels have been found in certain breast cancer cell lines [Plowman, G. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750], but little is known about the expression or the clinical significance of ErbB-4 receptors in the diagnosis and prognosis of human breast cancer.
To investigate the biological significance of ErbB-4 in human breast cancer, we used molecular targeting of the ErbB-4 mRNA by ribozymes. We describe the generation of three ribozymes (Rz6, Rz21, Rz29) targeted to specific sites within the ErbB-4 mRNA open reading frame. We demonstrate that all three ErbB-4 ribozymes cleave ErbB-4 mRNA precisely and efficiently under physiological conditions in this cell free system. We also illustrate the intracellular efficacy and specificity of the ErbB-4 ribozymes in a model system (32D cell system). 32D cells are a murine hematopoietic IL3-dependent cell line that does not express detectable levels of endogenous EGF-family receptors. Overexpression of ErbB-4 receptors in 32D cells (32D/ErbB-4) abrogated IL-3-dependence by stimulation with NRG. We show that two of the ErbB-4 ribozymes (Rz6 and Rz29) were able to down-regulate ErbB-4 expression and were capable of abolishing the neuregulin-induced mitogenic effect in 32D/ErbB-4 cells. These results demonstrate that ribozyme Rz29 and Rz6 are biologically functional ribozymes.
Therefore, this invention relates to ribozymes, or enzymatic RNA molecules, directed to cleave mRNA species encoding specific sites in ErbB-4. In particular, applicants describe the selection and function of ribozymes capable of cleaving this RNA and their use to reduce activity of ErbB-4 in various tissues to treat the diseases discussed herein, more particularly, breast cancer. Such ribozymes are also useful for diagnostic applications.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript and efficient cleavage has been achieved in vitro [Jefferies, et al. (1989) Nucleic Acid Res. 17:1371].
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic protion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action [Woolf, T. M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:7305-7309]. Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site. Consequently, the ribozyme agent will only affect cells expressing that particular gene, and will not be toxic to normal tissues.
The invention can be used to treat cancer or pre-neoplastic conditions. Two preferred administration protocols can be used, either in vivo administration to reduce the tumor burden, or ex vivo administration to eradicate transformed cells from tissues such as bone marrow prior to implantation.
Thus, in the first aspect the invention features an enzymatic RNA molecule (or ribozyme) which cleaves mRNA associated with development or maintenance of cancer, e.g. those mRNAs produced from the gene ErbB4 including mRNA targets disclosed in Table 1.
Hammerhead ribozymes (Rz) targeted to sites within ErbB-4 mRNA described in Table 1 were generated. These ErbB-4 ribozymes (Rz6, Rz21, Rz29) effectively catalyzed the precise cleavage of ErbB-4 mRNA under physiological conditions in a cell-free system. One of these ribozymes, Rz29, down-regulated ErbB-4 receptor expression by as much as 65%, with a corresponding 10-fold decrease in ErbB-4 tyrosine phosphorylation in a 32D cell model system. Furthermore, expression of this functional ErbB-4 ribozyme in T47D and MCF-7 human breast carcinoma cells led to a down-regulation of endogenous of ErbB-4 expression and a reduction of anchorage-independent colony formation.
By xe2x80x9cenzymatic RNA moleculexe2x80x9d it is meant an RNA molecule which has complementarity in a substrate binding region to a specified mRNA target, and also has an anzymatic activity which is active to specifically cleave that mRNA. That is, the enzymatic RNA molecule is able to intermolecularly cleave mRNA and thereby inactivate a target mRNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA to allow the cleavage to occur. One hundered percent complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention.
Ribozymes that cleave the specified sites in ErbB4 RNAs represent a novel therapeutic approach for the treatment of tumors and other conditions where overexpression of ErbB-4 is causal such as childhood medulloblastoma [Gilbertson, R. J. et al. (1998) Cancer Res. 58:3932-3941]. Applicants show that ribozymes are able to inhibit the activity of ErbB4 and that the catalytic acitiviy of the ribozymes is required for their inhibitory effect. Those of ordinary skill in the art, will find that it is clear from the examples described that other ribozyems that cleave these sites in ErbB4 RNAs may be readily designed and are within the scope of this invention.
In a second aspect, the invention features a mammalian cell which includes an enzymatic RNA molecule as described above. Preferably, the mammalian cell is a human cell.
In a third aspect, the invention features an expression vector which includes nucleic acid encoding an enzymatic RNA molecule described above, located in the vector, e.g., in a manner which allows expression of that enzymatic RNA molecule within a mammalian cell.
In a fourth aspect, the invention features a method for treatment of breast cancer by administering to a patient an enzymatic RNA molecule as described above.
The enzymatic RNA molecules of this invention can be used to treat human breast cancer. Such treatment can also be extended to other related genes in nonhuman primates. Affected animals can be treated at the time of cancer detection or in a prophylactic manner. This timing of treatment will reduce the number of affected cells.